Mammals contain at least three major, noninterconvertible isozymes of pyruvate kinase (EC 2.7.1.40). The isozymes have major kinetic and physical differences that enable them to serve specific physiological functions in the tissues where they are found but are similar enough that natural isozyme hybrids occur when a cell synthesizes two or more isozymes simultaneously. Type K is the main isozyme of all early fetal tissue and rapidly growing tumor cells but also occurs in varying amounts in most or all adult tissues. Type M predominates in skeletal muscle, heart, and brain, while type L is found in liver, kidney, and intestinal mucosa. We hope to study the structural properties of erythrocyte pyruvate kinase, which is immunologically similar to type L but electrophoretically distinguishable from it, and of type K, which is immunologically cross-reactive with type M but electrophoretically and kinetically distinguishable from the latter. We will analyze the effects of known or suspected modulators on the catalytic and physical properties of the purified enzymes and will compare their primary structures by peptide mapping. Shifts in isozymic synthesis will be determined in fetal red blood cells by electrophoretic analysis, and radioisotope incorporation followed by specific immunoprecipitation will be used to measure synthetic and degradative rates of the isozymes in normal and regenerating liver. In addition, we intend to assess the importance of phosphorylation and of specific ligand binding in regulating the catalytic activity of pyruvate kinases. Deficiencies in erythrocyte pyruvate kinase are associated with an important class of hereditary, nonspherocytic hemolytic anemia, while inhibition of pyruvate kinase by high levels of phenylalanine may be important in the brain damage resulting in untreated cases of phenylketonuria. Through the studies described above, we hope to better define the roles of the different isozymes in normal carbohydrate metabolism as well as in clinically important abnormal conditions.